Time-Restricted Feeding Prevents Obesity and Metabolic Syndrome in Mice Lacking a Circadian Clock

Abstract

Increased susceptibility of circadian clock mutant mice to metabolic diseases has led to the idea that a molecular clock is necessary for metabolic homeostasis. However, these mice often lack a normal feeding-fasting cycle. We tested whether time-restricted feeding (TRF) could prevent obesity and metabolic syndrome in whole-body Cry1;Cry2 and in liver-specific Bmal1 and Rev-erbα/β knockout mice. When provided access to food ad libitum, these mice rapidly gained weight and showed genotype-specific metabolic defects. However, when fed the same diet under TRF (food access restricted to 10 hr during the dark phase) they were protected from excessive weight gain and metabolic diseases. Transcriptome and metabolome analyses showed that TRF reduced the accumulation of hepatic lipids and enhanced cellular defenses against metabolic stress. These results suggest that the circadian clock maintains metabolic homeostasis by sustaining daily rhythms in feeding and fasting and by maintaining balance between nutrient and cellular stress responses.

Keywords: cell response to stress; circadian clock; circadian clock mutant mice; feeding-fasting rhythms; hepatic metabolomics; hepatic transcriptomics; metabolic diseases; metabolic homeostasis; metabolic syndrome; time-restricted feeding.

Figure 1:. Time-restricted feeding protects clock mutant mice from body weight gain on HFD.

A-G-M. Schematic of the experimental design depicting the 12 mice cohorts that were studied and the timing of food access relative to a 24h day. B-H-N. Evolution of body weight in (B) Bmal1^LKO (n=17-23/group), (H) Rev-erbα/β^LDKO (n=16-19/group), (N) CDKO (n=24/group). Multiple t test with unequal SD and Holm-Sidak correction for multiple comparisons, * p<0.05. C-I-O. Cumulative food consumption (kcal) in (C) Bmal1^LKO (n=17-23/group), (I) Rev-erbα/β^LDKO (n=16-19/group), (O) CDKO (n=24/group). D-J-P. Body composition (g) in (D) Bmal1^LKO and littermate controls (n=4-5/group), (J) Rev-erbα/β^LDKO and littermate controls (n=8-11/group), (P) CDKO and littermate controls (n=8-9/group) after 12 weeks on FA or FT. E-K-Q. Total activity during 3 days on TRF in (E) Bmal1^LKO mice (n=4/group), (K) Rev-erbα/β^LDKO (n=4/group) and (Q) CDKO (n=4/group). 1 way ANOVA with Holm-Sidak correction for multiple comparisons, * p<0.05. F-L-R. Running time on a treadmill during a run-to-exhaustion assay. Each individual dot represents one mouse. Unpaired t test, * p<0.05.

Figure 2:. Time-restricted feeding drives diurnal rhythms in fuel utilization.

A-E-I. Respiratory exchange ratio (RER) from metabolic cages recordings in (A) Bmal1^LKO (n=4/group, 5.5 days of recording), (E) Rev-erbα/β^LDKO (n=4/group, 4.5 days recording), (I) CDKO (n=4/group, 4.5 days recording). B-F-J. Corresponding VO2 (normalized to body weight) in (B) Bmal1^LKO (n=4/group), (F) Rev-erbα/β^LDKO (n=4/group) and (J) CDKO (n=4/group). C-G-K. Corresponding activity (Xt) in (C) Bmal1^LKO (n=4/group), (G) Rev-erbα/β^LDKO (n=4/group) and (K) CDKO (n=4/group). D-H-L. Corresponding food consumption (g) in (D) Bmal1^LKO (n=4/group), (H) Rev-erbα/β^LDKO (n=4/group) and (L) CDKO (n=4/group).

Figure 3:. TRF prevents whole-body fat accumulation and hyperlipidemia in clock deficient mice.

A-B-C. Relative fat mass as a percent of body weight in (A) Bmal1^LKO and littermate controls (n=4-5/group), (B) Rev-erbα/β^LDKO and littermate controls (n=8-11/group), (C) CDKO and littermate controls (n=8-9/group). Unpaired t-test, * p<0.05, ** p<0.01, *** p<0.001. D-E-F. Representative pictures of H&E staining of sections of (D) epididimal WAT, (E) BAT and (F) liver in the indicated genotype and feeding group. G-H. Serum leptin (G) and adiponectin (H) levels in Bmal1^LKO (n=10/group), Rev-erbα/β^LDKO (n=8/group), and CDKO (n=12/group). Unpaired t-test, * p<0.05, ** p<0.01, *** p<0.001. I-J-K. Liver triglyceride levels in (I) Bmal1^LKO and littermate controls (n=12/group), (J) Rev-erbα/β^LDKO and littermate controls (n=16/group), and (K) CDKO and littermate controls (n=8/group). Unpaired t-test, * p<0.05, ** p<0.01, *** p<0.001. L-M-N. Serum triglyceride (TG) levels in (L) Bmal1^LKO and littermate controls (n=8-10/group), (M) Rev-erbα/β^LDKO and littermate controls (n=8-12/group), and (N) CDKO and littermate controls (n=12/group). Unpaired t-test, * p<0.05, ** p<0.01, *** p<0.001. O-P-Q. Serum cholesterol levels in (O) Bmal1^LKO and littermate controls (n=8-10/group), (P) Rev-erbα/β^LDKO and littermate controls (n=8-12/group), and (Q) CDKO and littermate controls (n=12/group). Unpaired t-test, * p<0.05, ** p<0.01, *** p<0.001.

Figure 4:. Clock deficient mice on time-restricted feeding are protected from glucose intolerance and insulin resistance.

A-B-C. Glucose tolerance test (ip-GTT) in (A) Bmal1^LKO and littermate controls (n=8-10/group), (B) Rev-erbα/β^LDKO and littermate controls (n=8-12/group), and (C) CDKO and littermate controls (n=6-8/group). Quantification of the AUC above baseline is shown in the insert. Unpaired t-test, * p<0.05, ** p<0.01, *** p<0.001. D-E-F. Serum glucose levels in fasted (ZT22-ZT36) and refed mice (1h after IP injection of glucose (1mg/g BW) at ZT36) in (A) Bmal1^LKO and littermate controls (n=4-6/group), (B) Rev-erbα/β^LDKO and littermate controls (n=4-6/group), and (C) CDKO and littermate controls (n=4-6/group). G-H-I. Corresponding serum insulin concentration.

Figure 5:. Hepatic metabolomic footprint in clock-deficient mice on TRF.

A-B-C-D. Principal component analysis within genotype of metabolomics data obtained from liver extracts in (A) WT (n=6/group), (B) Bmal1^LKO (n=6/group), (C) Rev-erbα/β^LDKO (n=8/group), and (D) CDKO (n=8/group). E. Top significant pathways obtained from comparing significantly modulated metabolites between Bmal1^LKO on FA versus FT (purple) and Rev-erbα/β^LDKO on FA versus FT (green). F. Heatmap representation of the relative expression of indicated fatty acids in Bmal1^LKO on FA and FT and Rev-erbα/β^LDKO on FA and FT. G. Relative levels of medium chain fatty acid in the lover of Bmal1^LKO, Rev-erbα/β^LDKO and CDKO on FA and FT as indicated. H. Schematic representation of TCA cycle intermediates and their connection to glucose and lipid metabolic pathway showing liver metabolites that are significantly higher in FT (blue) or FA (red). Bar graphs of the relative levels of indicated metabolites levels are shown below. I. Serum β-Hydroxybutyrate levels in fasted (ZT22-ZT36) and refed mice (1h after IP injection of glucose (1mg/g BW) at ZT36) in Bmal1^LKO and littermate controls (n=4-6/group), Rev-erbα/β^LDKO and littermate controls (n=4-6/group), and CDKO and littermate controls (n=4-6/group).

Figure 6:. Liver transcriptomics unravel similarities and differences between clock mutants on TRF.

A- PCA plot of liver transcriptome data. B-C. Schematic depicting the number of significant genes between each group as indicated. D- Expression level (normalized read count (log2)) of some genes involved in fatty acid oxidation that are significantly lower in Bmal1^LKO-FA versus WT-FA. E- Expression level (normalized read count (log2)) of some genes involved in cholesterol metabolism that are significantly lower in Rev-erbα/β^LDKO versus WT-FA. F. Expression level (normalized read count (log2)) of enzymes involved in lipid metabolism positioned on a schematic lipid pathway representation as well as expression level of the master lipid regulators Pparα, Pparγ and Srebf1. G. Heatmap representation of the expression levels of 366 cycling genes in WT-FA and 172 cycling genes in WT-FT and their corresponding expression in clock deficient mice as indicated. H. Heatmap representation of the expression level of 43 cycling genes in WT-FA and WT-FT and their corresponding expression in clock deficient mice as indicated. I. Examples of cycling genes in WT. B,D,E: differentially expressed genes with adjusted p value<0.05. C,F: differentially expressed genes with feeding_adjusted p value<0.01 and adjusted p interaction > 0.1.

Figure 7:. Liver function is preserved in TRF and diurnal rhythms in nutrient sensing pathway is preserved in all clock mutants on TRF.

A. Pathway enrichment analysis results of 2142 genes that are significantly higher in TRF. Related pathways are colored similarly. B. Heatmap representation of the expression levels of the significant genes in TRF belonging to DNA metabolism and transcription (83 genes, blue bars in 7.A.) (ii) RNA processing (114 genes, yellow bars in 7.A.), (iii) Protein metabolism (112 genes, dark grey bars in 7.A.), (iv) Protein folding and unfolded protein response (94 genes, light grey bars in 7.A.), and (v) Defense response and coagulation (113 genes, red bars in 7.A.) and selected individual expression profile (below). C-D. Representative western blot and graphical representation of the quantification (ImageJ) of the temporal expression profile of (C) phospho-ERK/ERK, GCK and active SREBP (cleaved form) and (D) the phosphorylated ribosomal protein S6 (P-S6), in the liver of clock deficient mice and control in FA versus FT as indicated. E. Diurnal levels of the metabolite tryptophan in the liver of clock deficient mice and control in FA versus FT as indicated. F. Diurnal levels of Gabarapl1 expression in the liver of clock deficient mice and control in FA versus FT as indicated.